Molecular Diagnostics 2 Flashcards
(95 cards)
What does ASO stand for?
Allele-Specific Oligonucleotide Hybridization
What is the point of ASO hybridization?
to detect small, sequence variants. Usually single base, but can be very small like a three base mutation. Normally point mutations
When doing a ASO hybridization you have to know what about mutation
where it is
ASO hybridization can be useful for screening for ____ mutations
frequent
for ASO you need how many probes are needed?
one for wild type
one for mutation
What do you do first to amplify the DNA for ASO
PCR amplify the product
Is there separation on basis of size for ASO?
No
What are you looking at in ASO hybridization, if not size
if there is a mutation there or not
What is a dot blot?
How you see if mutation is there in ASO hybridization
dark blot on normal probe tells you want
pt has the normal alelle
Why are short probes used for ASO hybridization
for specificity
Sickle cell disease is caused by what kind of muatation
single point mutation, A to T
E6V means what
Glutamate to valine
What is the specific mutation for sickle cell disease
E6V
How many probs d you need to ASO to detect sickle cell disease
2 - one for normal one for mutatatn
The probes for ASO are very small and also have what characteristic
Very specific - so the mutant probe will only bind to mutant and normal will only bind to normal
What does PCR stand for
polymerase chain reaction
What does PCR do at its basic level
exponentially amplify DNA
PCR is a _____ reaction that doubles the amount of DNA
cyclical
PCR uses what kind of polymerase
DNA polymerase
What are 2 of the negative sides of PCR?
need at least some sequence info to make primers
susceptible to contaminiation due to high sensitivity
What polymerase is most often used for PCR?
taq polymerase
PCR does DNA synthesis by a thermostable:
DNA polymerase
What is one downside of taq?
no proofreading
